Figure 1. Requirement for TGF-β in the Maintenance of IL-17 Expression by Th17-Polarized Cells.

(A) FACS-sorted naive CD4⁺ T cells from OT-II TCR transgenic mice were cultured with irradiated Il12b^−/− (IL-12p40-deficient) splenic feeder cells and 5 μg/ml OVAp for 7 days under Th17-polarizing conditions (TGF-β, 5 ng/ml; IL-6, 20 ng/ml; anti-IFN-γ, 10 μg/ml; anti-IL-4, 10 μg/ml), then stained intracellularly for IL-17A and IFN-γ after PMA/ionomycin activation for 5 hr in the presence of monensin (1° culture; left panel). Cells were harvested and restimulated with irradiated Il12b^−/− splenic feeder cells in the presence of indicated cytokine(s) at the same doses or, in the case of IL-23 and IL-12, at 1 ng/ml, with anti-IFN-γ, anti-IL-4, and OVAp for an additional two rounds (7 days each) and stained intracelluarly for IL-17A and IFN-γ after PMA-ionomycin activation (post 3° culture; right panels).

(B) Ifng^{Thy1.1/Thy1.1} OT-II naive CD4⁺ T cells were cultured with irradiated Il12b^−/− splenic feeder cells under Th17-polarizing conditions for 6 days and a fraction of the recovered cells were stained intracellularly for IL-17A and IFN-γ after PMA-ionomycin activation (left panel). A second fraction of recovered CD4⁺ T cells were activated for 5 hr with OVAp and Il12b^−/− feeder cells, and Thy1.1⁺ (IFN-γ⁺) cells were depleted by magnetic sorting. Thy1.1 depletion was confirmed by surface staining for Thy1.1 (middle panel), and the isolated Thy1.1- cells were cultured with Il12b^−/− feeder cells and OVAp and either TGF-β plus IL-6 or IL-23 in the presence of anti-IFNγ plus anti-IL-4 for three additional rounds (right panels), then stained intracellulary for IL-17A and IFN-γ after PMA-ionomycin activation. Numbers in each quadrant indicate percentages of total CD4⁺ T cells. All data are representative of at least three independent experiments.